ORIGINAL ARTICLE

Photodynamic inactivation of planktonic

cultures and Streptococcus mutans
biofilms for prevention of white spot
lesions during orthodontic treatment: An
in vitro investigation
^
Maria Angela Lacerda Rangel Esper,a Juliana Campos Junqueira,b Adjaci Fernandes Uchoa,c
Eduardo Bresciani,a Alessandra Nara de Souza Rastelli,a Ricardo Scarparo Navarro,c and
Sergio Eduardo de Paiva Gonçalvesa
S~ao Paulo, Brazil

Introduction: This study evaluated the efficacy of photodynamic inactivation (PDI) with hematoporphyrin IX (H)
and modified hematoporphyrin IX (MH) at 10 mmol/L, using a blue light-emitting diode (LED), fluence of 75 J/cm,2
over planktonic cultures and biofilm of Streptococcus mutans (UA 159). Methods: Suspensions containing
107 cells/mL were tested under different experimental conditions: a) H and LED (H1L1), b) MH and LED
(MH1L1), c) only LED (P L1), d) only H (H1L ), e) only MH (MH1L ), and f) control group, no LED or
photosensitizer treatment (P L ). The study also evaluated the effect of PDI on S mutans biofilm on
metallic or ceramic brackets bonded on specimens of human teeth. The strains were seeded onto
Mitis salivarius–bacitracin–sacarose agar to determine the number of colony-forming units. Results: H and
MH under LED irradiation were effective on planktonic cultures (P \0.0001). H and MH (H1L1 and MH1L1)
caused a reduction of 3.80 and 6.78 log10 CFU/mL. PDI with the use of H or MH and LED exerted a strong
antimicrobial effect over S mutans showing 54% and 100% reduction, respectively. PDI on S mutans biofilm
on metallic and ceramic brackets with the use of H was not effective (P 5 0.0162, P 5 0.1669), however, MH
caused a significant reduction of 44% and 53% of the cell count on metallic and ceramic brackets, respectively
(P 5 0.0020, P 5 0.004). Conclusions: In vitro planktonic cultures with the use of H or MH and LED exerted
significant antimicrobial activity. No effect was observed on S mutans biofilm on either bracket type with the
use of H, MH showed better results, suggesting a promising use against dental caries and white spot lesions.
(Am J Orthod Dentofacial Orthop 2019;155:243-53)

A
lthough orthodontic treatment has advanced
with innovative material and techniques, some
a
Department of Restorative Dentistry, Institute of Science and Technology,
aspects still need further study, such as pain
S~ao Paulo State University, S~ao Paulo, Brazil. sensitivity reported by patients after orthodontic
b
Department of Biosciences and Oral Diagnosis, Institute of Science and Tech- movement1 and preventive methods to avoid white
nology, S~ao Paulo State University, S~ao Paulo, Brazil.
c
Department of Biomedical Engineering, Anhembi Morumbi University, S~ao
spot lesions and decay during orthodontic treatment
Paulo, Brazil. with the use of fixed appliances.2,3
All authors have completed and submitted the ICMJE Form for Disclosure of The placement of removable and/or fixed
Potential Conflicts of Interest, and none were reported.
^
Address correspondence to: Maria Angela Lacerda Rangel Esper, Department of
orthodontic appliances causes modification in the oral
Restorative Dentistry, Institute of Science and Tecnology, S~ao Paulo State ecosystem, increasing the number of cariogenic
University—UNESP, Avenida Engenheiro Francisco Jose Longo, 777, Jardim bacteria.4,5 It also favors the formation of biofilm and
S~ao Dimas, S~ao Jose dos Campos, SP, CEP 12245-000, Brazil; e-mail,
angela_esper@hotmail.com.
restricts salivary self-cleaning, and oral hygiene becomes
Submitted, August 2017; revised and accepted, March 2018. more difficult.6,7 An imbalance occurs between the
0889-5406/$36.00 processes of demineralization and remineralization,
Ó 2018 by the American Association of Orthodontists. All rights reserved.
https://doi.org/10.1016/j.ajodo.2018.03.027
increasing the risk of demineralization and decay and

243
244
the development of periodontal inflammation, with
notable changes in the microbiota. Many clinical
studies have shown that patients undergoing fixed
orthodontic treatment are more susceptible to
periodontal diseases and white spot lesions (WSLs),
regardless of the type of bracket used,8-11 especially if
the patient does not cooperate with oral hygiene.6,12-15
Demineralization around the brackets is an adverse
effect of great clinical relevance, despite modern
advances in the prevention of dental caries.6,16,17 It is
well known that WSLs can be seen as early as 4 weeks
after the start of treatment with fixed orthodontic
appliances,18,19 especially in the cervical region and
around the brackets,12 if preventive measures are not
established20 and depending on the host conditions
(highly acidic saliva, cariogenic diet).21
The usual methods to remove the oral microbial
biofilm are tooth brushing, use of dental flossing and
antiseptics. However, biofilm removal depends on
patient compliance, and the use of antiseptics could
produce drug-resistant microbes and lead to other
side-effects.22 It is necessary to highlight the importance
of diet orientation for the patients during orthodontic
treatment to prevent WSLs and tooth decay.23
Photodynamic therapy (PDT) has been applied in
many areas of medicine and dentistry with effective
results.24,25 Antimicrobial PDT or photodynamic
inactivation (PDI) might be an alternative for biofilm
control or removal, prevention of WSLs, dental caries,
and periodontal diseases26 with the use of a light source
(halogen lamps, laser, or light-emitting diode [LED]) to
activate a specific photosensitizer in the presence of
oxygen, producing reactive radicals that induce cell
death.27 The use of PDI to treat different oral infections
has important advantages over conventional therapeutic
treatments. It can promote effective antimicrobial
activity without the development of microbial resistance
and can be used repeated times without limitation of
total dose.27,28
There are many photosensitizers that can be used for
PDI, and the amount of singlet oxygen generated can
change for each photosensitizer or dye. This is called
the singlet oxygen quantum yield (QY), and the QY of
hematoporphyrin derivatives are high29 and an
advantage compared with other photosensitizers.
Hematoporphyrin IX is derived from protoporphyrin
and has been used as a photosensitizer for PDT.
However, its application is limited owing to its
aggregation and low solubility in a physiologic medium.
Modifications to the protoporphyrin IX molecule have
been made to increase the solubility in a physiologic
medium and stimulate its use in PDT. Thus, hydroxyl
groups have been inserted in the vinylic positions 31,
February 2019  Vol 155  Issue 2 American Journal of Orthodontics and Dentofacial Orthopedics
Lacerda Rangel Esper et al
81, 133, and 173 to produce modified hematoporphyrin
IX in both acid and basic media and to potentialize
global income.30,31
LEDs are commonly used in the dental office for light-
curing composite resins and recently for tooth whitening
and has many advantages compared with laser, such as
low cost and simplicity.32 The emission spectrum of the
blue LED matches the activation spectrum of the
photosensitizers used in the present study.33 There are
no papers in the literature describing the effect of PDI
with the use of hematoporphyrin IX or modified
hematoporphyrin IX and LED on planktonic cultures
and biofilms of Streptococcus mutans on human teeth
specimens bonded with metallic and ceramic brackets.
Therefore, the purpose of the present study was to
evaluate the effect of PDI with the use of
hematoporphyrin IX or modified hematoporphyrin IX
(experimental) photosensitizers irradiated by a blue LED
on the viability of planktonic cultures and biofilms of
S mutans on metallic and ceramic brackets. The null
hypothesis was that hematoporphyrin IX and modified
hematoporphyrin IX do not decrease the numbers of
CFU/mL on planktonic cultures and biofilms of S mutans.
MATERIAL AND METHODS
This study was submitted to the Institutional Review
Board of S~ao Paulo State University—UNESP (CONEP;
CAAE: 44832215.0.0000.0077; IRB: 1.190.860) because
of the use of human teeth (third molars) indicated for
extraction.
Specimen preparation
Eighty cylinders of 6.0 mm 3 2.15 mm (n 5 10 for
each group) were obtained with the use of a diamond
trephine drill from 30 recently extracted human
impacted third molar teeth (2 or 3 specimens from
each tooth), without any caries or visible defects and
kept in 0.1% thymol. After cutting, the cylinders were
polished (DP-10; Panambra, S~ao Paulo, Brazil) with
the use of silicon-carbide abrasive paper (grit #1200;
Extec Corp, Enfield, Conn) for 30 seconds at 300 rpm.
After polishing, the specimens were immersed in an
ultrasonic bath for 10 minutes (Ultrasonic Cleaner;
Odontobras, Ribeir~ao Preto, Brazil). The final specimens
measured 6 mm diameter and 2 mm height as verified by
means of a digital caliper (Starrett, S~ao Paulo, Brazil).
Before bonding technique, the buccal surface of each
specimen was cleansed and polished by means of water
and nonfluoridated pumice mixture with the use of a
low-speed handpiece for 6 seconds. Each specimen
was etched with the use of 37% phosphoric acid gel
(Gel Etch; 3M Unitek, Monrovia, Calif) for 30 seconds,
Lacerda Rangel Esper et al
Fig 1. Absorption spectrum (nm) of hematoporphyrin IX and modified hematoporphyrin IX
photosensitizers.
rinsed for 10 seconds, and air dried for 5 seconds. A thin
uniform layer of sealant (Orthoprimer, ref 85.01.016;
Morelli, Sorocaba, Brazil) was brushed on the etched
enamel surface and on the metallic and ceramic bracket
base. The paste (Orthobond, ref 85.01.015; Morelli) was
placed onto the bracket base with the use of a spatula for
composite resin. An explorer probe was used to seat the
bracket on the enamel surface with a consonant force,
and excessive adhesive was removed. The composite
resin was light cured with the same visible light-curing
unit used for PDI at 1200 mW/cm2 (Emitter C; Schuster,
Santa Maria, Brazil) for 40 seconds (10 s-on the distal,
10 seconds on the mesial, 10 seconds on the incisal,
and 10 seconds on the gingival surface of the bracket).
In this study, a 0.022-inch inferior incisor metallic (ref
10.30.209; Morelli) and ceramic (ref 10.18.012; Morelli)
brackets were bonded on the enamel surface. After
bonding, the specimens were kept in deionized water
at 37 C. Before the experiments, described below, spec-
imens with bonded brackets and a metallic supports
were sterilized by means of autoclaving in Falcon tubes
containing deionized water for 30 minutes at 135 C.
Photosensitizers and light source
Hematoporphyrin IX (C36H42N4O6) and modified
hematoporphyrin IX (C50H74N8O2) dihydrochloride
synthesized at the Institute of Chemistry (S~ao Paulo Uni-
versity—USP, S~ao Paulo, Brazil) were used at
concentrations of 10 mmol/L for the sensitization of
American Journal of Orthodontics and Dentofacial Orthopedics
245
S mutans. The photosensitizer solutions were prepared
by dissolving the dye in dimethyl sulfoxide (DMSO;
Merck) and 0.85% sodium chloride. After filtration
through a sterile 0.20-mm Millipore membrane (GVS,
Sanford, Me), the photosensitizer solutions were stored
in the absence of light. Figure 1 shows the absorption
spectrum for hematoporphyrin IX and modified
hematoporphyrin IX, and Figure 2 their chemical
structures.
The light source used was a wireless blue LED
(Emitter C; Schuster) at a wavelength of 420-480 nm,
power output of 625 mW, and an illuminated area of
0.5 cm2. A fluence of 75 J/cm2 (energy of 37.5 J and
irradiation time of 60 s) and a fluence rate of
1250 mW/cm2 were used. Emission spectrum of the
blue LED is shown in Figure 3.
Microorganism and production of the biofilms
The planktonic culture methodology was as
described by Costa et al34 and the biofilm formation as
described by Pereira et al.26 The reference strain
(UA 159) of S mutans maintained in our laboratory stock
collection was used in this study. Standard suspension
containing 107 cells/mL was prepared. For this
purpose, S mutans was seeded onto brain heart infusion
(BHI) and incubated for 24 hours at 37 C (61 C)
under microaerophilic conditions. After incubation, the
microorganisms were cultured in 7.5 mL BHI broth
(Himedia) for 24 hours at 37 C (61 C) under
February 2019  Vol 155  Issue 2
246 Lacerda Rangel Esper et al

Fig 2. Chemical structure of hematoporphyrin IX (left) and modified hematoporphyrin IX (right)

photosensitizers.

Fig 3. Emission spectrum of the blue LED (Emitter C; Schuster, Santa Maria, Brazil).

microaerophilic conditions. All incubations were carried
out at 37 C (61 C) in 5% CO2. The bacterial cultures
were then centrifuged at 4700g for 5 minutes, the
supernatant discarded, and the pellet washed with the
use of 5 mL sterile saline solution (0.85% sodium
chloride). This procedure was repeated, and the pellet
was resuspended in 5 mL sterile physiologic solution.
The number of cells in each suspension was measured
in a spectrophotometer (B582; Micronal, S~ao Paulo,
Brazil) at a wavelength of 398 nm, and an optical density
of 0.560 was reached. These parameters were previously
established by means of a standard curve with
colony-forming units (CFU) versus absorbance.
The biofilms were grown over sterile metallic
or ceramic brackets bonded on specimens of human
teeth. The specimens were placed in 24-well plates
(Costar Corning, New York, NY), suspended in
each well by means of a metallic support (Figs 4,

February 2019  Vol 155  Issue 2 American Journal of Orthodontics and Dentofacial Orthopedics
Lacerda Rangel Esper et al
Fig 4. A, Metallic support made with stainless steel orthodontic wire for specimens support.
B, Specimen placement in a 24-well plate. C, Biofilm formation, specimens were suspended, prevent-
ing them from touching the well bottoms.
A and B) made with stainless steel orthodontic wire
(0.016 inch 3 0.016 inch, ref 55.02; Morelli), with
2 mL sterile broth and were inoculated with 0.1 mL
S mutans standard suspensions. The metallic supports
were made to suspend the specimens and prevent
them from touching the well bottoms, which had more
accumulation of biofilm, to simulate the oral cavity
(Fig 4, C). After 24 hours, the broth was aspirated and
refreshed. The plates were incubated for 48 hours.
For each photosensitizer, the assays were submitted
to 4 experimental conditions (n 5 10 each), as described
in the Table, at 2 different times.
In vitro photosensitization of planktonic cultures
According to the experimental conditions described,
0.1 mL bacterial suspension was added to each well of
sterile 96-well flat-bottom microtiter plates (Costar
Corning). Next, 0.1 mL photosensitizer was added for
groups H1L1, MH1L1, H1L , and MH1L , whereas
0.1 mL sterile physiologic solution was added for groups
P L1 and P L . The plates were shaken for 5 minutes
before irradiation in an orbital shaker (Solab, Piracicaba,
Brazil). After that, the well content of groups H1L1,
MH1L1, P L1 was irradiated according to the
protocol described above. The distance between the light
source and the bacterial cells was 6 mm. Irradiation
was performed under aseptic conditions in a laminar
flow hood in the dark. The plates were covered with a
sterilized matte black screen with an orifice whose
diameter corresponded to the size of the well entrance
American Journal of Orthodontics and Dentofacial Orthopedics
247
Table. Experimental groups
Group Experimental condition
H1L1 LED irradiation with hematoporphyrin IX as
photosensitizer
H1L Hematoporphyrin IX only
MH1L1 LED irradiation with modified hematoporphyrin IX as
photosensitizer
MH1L Modified hematoporphyrin IX only
P L1 LED irradiation only
P L No LED irradiation or photosensitizer (control group)
to prevent the spreading of light to neighboring wells.
After irradiation, serial dilutions were prepared, and
0.1 mL aliquots of each dilution were seeded in duplicate
onto Mitis salivarius–bacitracin–sacarose (MSBS) agar
plates and incubated for 48 hours at 37 C (61 C) under
microaerophilic conditions. After incubation, the
number of CFU/mL was determined.
In vitro photosensitization of biofilms
After 48 hours of incubation, the specimens
containing the biofilms were aseptically washed twice
in sterile 24-well flat-bottom microtiter plates with
sterile physiologic solution (0.85% NaCl) to remove
loosely bound bacteria. The specimens were then
placed, without the metallic supports, in sterile 96-well
flat-bottom microtiter plates. According to the
experimental conditions described in the Table,
0.15 mL photosensitizer was added for groups H1L1,
H1L , MH1L1, and MH1L and 0.15 mL physiologic
February 2019  Vol 155  Issue 2
248
Fig 5. Samples of Mitis salivarius–bacitracin–sacarose agar plates and colony-forming units as result
of bacteriology.
solution (0.85% NaCl) was added for groups P L1 and
P L . The plates were left in the dark and shaken for
5 minutes before irradiation with the use of an orbital
shaker (Solab). The biofilms of groups P L1, H1L1,
and MH1L1 were then irradiated according to the
protocol described above. The distance between the light
source and the bacterial cells and biofilms was 6 mm.
Irradiation was performed under aseptic conditions in a
laminar flow hood in the dark. The plates were covered
with a sterile matte black screen with an orifice whose
diameter corresponded to the size of the well opening
to prevent the spreading of light to neighboring wells.
After PDI, the specimens were washed, placed in tubes
with 10 mL sterile physiologic solution (0.85% NaCl)
and sonicated (Sonoplus HD 2200, 50 W; Bandelin
Eletronic, Berlin, Germany) for 30 seconds to disperse
the biofilms. Tenfold serial dilutions were carried out,
and 0.1 mL aliquots of each dilution were seeded in
duplicate onto MSBS agar plates and incubated for
48 hours at 37 C (61 C) under microaerophilic condi-
tions. After incubation, the number of CFU/mL was
determined (Fig 5).
February 2019  Vol 155  Issue 2 American Journal of Orthodontics and Dentofacial Orthopedics
Lacerda Rangel Esper et al
Statistical analyses
The results were log transformed and statistical
analyses were carried out with the use of the Graphpad
Prism 6.0 program (Graphpad Software, San Diego,
Calif). One-way analysis of variance and the Tukey test
were used. A P value of \0.05 was considered to
indicate a statistically significant difference.
RESULTS
Figures 6-11 present the mean values and standard
deviations of the number of CFU/mL (log10) obtained
for the different treatment groups studied.
The susceptibility of planktonic cultures of S mutans
to PDI with the use of hematoporphyrin IX and modified
hematoporphyrin IX (Figs 6 and 7) showed a reduction of
3.80 and 6.78 log10 CFU/mL for groups H1L1 and
MH1L1, respectively, compared with the control group
(P L ; P \0.0001). Groups H1L , MH1L , and
P L1 produced no reduction in the numbers of
CFU/mL of S mutans compared with the control group
(P L ).
Lacerda Rangel Esper et al
Fig 6. Mean values and standard deviations of the
number of CFU/mL (log10) obtained with the use of
PDI on planktonic cultures of S mutans mediated
by hematoporphyrin IX at 10 mmol/L and blue LED
(P \0.0001). Different letters indicate statistical
differences between groups.
Fig 7. Mean values and standard deviations of the
number of CFU/mL (log10) obtained with the use of PDI
on planktonic cultures of S mutans mediated by
modified hematoporphyrin IX at 10 mmol/L and blue
LED (P \0.0001). Different letters indicate statistical
differences between groups.
The biofilm assays for PDI with the use of
hematoporphyrin IX (H1L1, H1L , and P L1)
produced no reduction in the numbers of CFU/mL of
S mutans on metallic and ceramic brackets compared
with the control group (P L ; P 5 0.0162 and
P 5 0.1669, respectively), indicating that this photosen-
sitizer was not effective for the species studied (Figs 8
and 9). However, modified hematoporphyrin IX
(MH1L1) showed a reduction of 2.43 and 3.60
American Journal of Orthodontics and Dentofacial Orthopedics
249
Fig 8. Mean values and standard deviations of the
number of CFU/mL (log10) obtained with the use of PDI
on S mutans biofilms on metallic brackets mediated by
hematoporphyrin IX at 10 mmol/L and blue LED
(P 5 0.0162). Different letters indicate statistical
differences between groups.
Fig 9. Mean values and standard deviations of the
number of CFU/mL (log10) obtained with the use of PDI
on S mutans biofilms on ceramic brackets mediated by
hematoporphyrin IX at 10 mmol/L and blue LED
(P 5 0.1669). Different letters indicate statistical
differences between groups.
log10 CFU/mL on metallic and ceramic brackets
compared with the control group (P L ; P 5 0.0020
and P 5 0.0004, respectively), indicating that
MH associated with blue LED had a bactericidal
effect on S mutans biofilm (Figs 10 and 11). Groups
MH1L and P L1 showed no reduction of S mutans
compared with the control group (P L )
February 2019  Vol 155  Issue 2
250
Fig 10. Mean values and standard deviation of the
number of CFU/mL (log10) obtained with the use of PDI
on S mutans biofilms on metallic brackets mediated by
modified hematoporphyrin IX at 10 mmol/L and blue
LED (P 5 0.0020). Different letters indicate statistical
differences between groups.
DISCUSSION
Many techniques have been studied for the removal
or control of dental biofilm, which is one of the most
important etiologic agents for many oral diseases,
including dental caries, but with limited success.
Therefore, alternate techniques that do not depend on
patient compliance are required to prevent WSLs,
caries,22 and periodontal disease in orthodontic
patients.35
PDI is an alternate method to overcome the microbial
resistance of antibiotic drugs and chemical agents. PDI
combines an appropriate photosensitizer and light
source that is minimally invasive and nontoxic to
control the formation of oral biofilm.22,36 There are
some advantages of PDI over conventional
antimicrobial agents, such as the killing of the target
microorganisms being quick, depending on the
parameters used, the lack of resistance by the target
organisms, and that the antimicrobial effects can be
localized to a specific site, avoiding disruption of
normal microflora in other regions.27,28,37,38
The success of PDI is based on factors such as
type, concentration, preirradiation time of the photo-
sensitizer, physiologic condition of the microorganisms
studied, the light source, and the doses used.39
Therefore, in vitro investigation with the use of a
planktonic model is a major step for guiding in vivo pro-
tocols and parameters for more detailed future studies
when considering illumination aspects (related to the
light sources) and concentration (related to the dyes).40
The in vitro planktonic essays are also essential to
February 2019  Vol 155  Issue 2 American Journal of Orthodontics and Dentofacial Orthopedics
Lacerda Rangel Esper et al
Fig 11. Mean values and standard deviation of the
number of CFU/mL (log10) obtained with the use of PDI
of S mutans biofilms on ceramic brackets mediated by
modified hematoporphyrin IX at 10 mmol/L and blue
LED (P 5 0.0004). Different letters indicate statistical
differences between groups.
determine the efficacy of potential antimicrobial agents.
Most methods for evaluating antimicrobial activity of
new antimicrobial candidates are based on determining
cell viability in microbial suspensions.41 According to the
American Society of Microbiology, a CFU reduction of 3
log10 is necessary to use the term “antimicrobial.”42
Tests on planktonic or biofilm cultures may be per-
formed when carrying out microbiologic studies
in vitro. In planktonic cultures, microorganisms are
free or in small groups and there is an abundant avail-
ability of photosensitizer molecules. However, in biofilm
communities a more complex organization is observed,
forming a 3-dimensional structure with multiple cell
layers. It also occurs in the extracellular polysaccharide
matrix, with the incorporation of compounds that favor
intercellular adhesion, providing an increased resistance
to chemical procedures or antimicrobial compounds.
Therefore, similar PDI protocols can result in different
death rates in experiments on planktonic cultures
compared with biofilms.43
To establish a future clinical treatment protocol with
the use of PDI, there is a need for an evaluation of the
efficiency and definition of proper parameters. First,
in vitro experiments on planktonic cultures must be
carried out. If these experiments are not effective, this
treatment technique is likely to be unsuccessful. But
even if effectiveness is achieved in cell cultures, it is
not necessarily achieved in vivo. However, this sequence
of experiments, in vitro and later in vivo, is essential for
development in the biomedical area to ensure the safety
and efficacy of clinical protocols.43
Lacerda Rangel Esper et al
The present study showed an interesting result with
hematoporphyrin IX and modified hematoporphyrin IX,
killing 54% and 100% (P \0.0001) of S mutans in
planktonic culture, respectively. In addition, no
reduction of S mutans biofilms was observed with the
use of hematoporphyrin IX on both types of brackets.
However, modified hematoporphyrin IX showed a
significant inactivation of S mutans biofilms, with
44% and 53% survival rate (P 5 0.0020 and
P 5 0.0004), respectively, seen on metallic and ceramic
brackets. This is the first study using hematoporphyrin IX
and modified hematoporphyrin IX as photosensitizers on
planktonic culture and biofilms of S mutans.
Hematoporphyrin IX and modified hematoporphyrin
IX absorb light at 400 nm, and the light source used in
the present study emits light at the range of
420-480 nm. The association between the dyes and light
resulted in cellular death, probably due to the generation
of reactive oxygen species. The emission spectrum of the
blue LED matches the activation spectrum of the
photosensitizers used in this study, which is a basic
requirement for PDI.33 Significant differences in PDI
with the use of modified hematoporphyrin IX were
observed. The results suggested that modified
hematoporphyrin IX was more effective than
hematoporphyrin IX in the biofilms experiments. The
null hypothesis was rejected for both photosensitizers
in the first part (planktonic culture) and accepted for
hematoporphyrin IX and rejected for modified
hematoporphyrin IX in the second part (biofilms).
Hematoporphyrin IX and modified hematoporphyrin
IX44,45 are derivatives of protoporphyrin, which
naturally occurs in hemoglobin, cytochrome C, and
other biologically relevant molecules. It has been
used as a photosensitizer for PDT. However, the
application is limited owing to aggregation and low
solubility in a physiologic medium. Modifications to
the hematoporphyrin IX molecule have been made
to increase the solubility of this potential drug in a
physiologic medium and to encourage its use in PDT.
An important strategy is to explore the reactivity of
vinyl groups to functionalize Pp IX with polar groups.
To this end hydroxyl groups have been inserted in the
vinylic positions 31 and 81 to produce
hematoporphyrin IX. The hematoporphyrin IX, in both
acid and basic media, polymerizes to form a mixture of
chromophores named Photofrin, which is the most
used drug in PDT. Further functionalization of
carboxylic groups has also been used for the
preparation of several derivatives.4,5 Because this
chemical compound is a hemoglobin derivative, it is
well metabolized and it does not have the
disadvantage of other compounds that may stain the
American Journal of Orthodontics and Dentofacial Orthopedics
251
dental structures, esthetic dental restorations, and even
esthetic brackets when used in dentistry.30,31
The light source used in this study was based on a
blue LED, which presents many advantages.27,33,46
Dentists could use their light-curing unit, which is based
on blue, to perform PDI in their offices. Some studies
have shown that the use of LED alone exerts little or
no antimicrobial activity.32,47
According to Reddy et al (2012),48 there are 2 basic
mechanisms that have been proposed regarding the
lethal damage caused to bacteria with the use of PDI.
One of them is related to the DNA damage and the other
to the damage caused to the cytoplasmic membrane
allowing leakage of cellular contents or inactivation of
membrane transport systems and enzymes. Breaks in
both single- and double-stranded DNA and the
disappearance of the plasmid supercoiled fraction
have been detected in both gram-positive and
gram-negative bacteria. The other potential causes of
cell death include the alteration of cytoplasmic
membrane proteins, the disturbance of cell wall
synthesis, and the appearance of a multilamellar
structure near the septum of dividing cells; loss of
potassium ions from the cells may also be a possible
method of bacterial death.
There is only one longitudinal blind randomized study
in the literature of the effect of PDI in patients
undergoing orthodontic treatment. The authors used
curcumin irradiated by blue LED, compared it with 2%
chlorhexidine varnish in adolescent patients during
orthodontic treatment, and studied periodontal
inflammation.49 They concluded that PDI represents a
promising approach for the prevention of gingivitis in
adolescents during fixed orthodontic treatment.
However, they reported that further investigations must
be carried out to clarify the role of PDI in preventing dis-
ease and to encourage studies in larger samples, as well
as studies investigating the parameters with the purpose
of reducing the light exposure time and optimizing this
new treatment technique in patients under high risk of
developing various pathologic conditions.
There are no previous studies in the literature on
using PDI for the prevention of WSLs in patients
undergoing orthodontic treatment. There are important
PDI studies in planktonic cultures and S mutans
biofilms22,26,34,35,50-54 with interesting inactivation
results, but they used other photosensitizers and
concentrations and different light sources, doses, and
irradiation times. The results of those studies
are somewhat adequate regarding microorganisms
reduction, but comparison with our data is impaired as
a result of the differences in methodology. The
previous data were important, however, for proposing
February 2019  Vol 155  Issue 2
252
more efficient and more user-friendly protocols. Our
study was based on some of those previous works,26,34
which used an LED wavelength range of 440-460 nm.
The same energy, but a different strain of S mutans
(UA 159), was used in the present study.
Currently, there is a wide variety of orthodontic
brackets (metallic, plastic and ceramic conventional,
metallic and ceramic self-ligating) that the orthodontist
may elect to use with their patients. However, owing to
increased demand for orthodontic treatment in adult
patients, there is also a high esthetic requirement by
the patients and therefore there is a significant increase
in the use of ceramic (polycrystalline alumina) and
plastic (polycarbonate) brackets.9 There are several
studies on cariogenic bacterial colonization and
periodontal changes in the different compositions and
bracket designs, as well as in metallic versus elastic
ligatures. The authors are unanimous in stating
that there is a significant increase of cariogenic
microorganisms after the installation of fixed
orthodontic appliances, regardless of the type of
brackets used. This factor may favor the appearance of
WSLs if there is no patient cooperation with oral hygiene
and diet orientation.7-11,19,23 Despite the focus of the
present study not being the type of brackets, there was
no significant difference in the number of CFU/mL of
S mutans between the 2 types of brackets, metallic
and ceramic, tested. These results are in accordance
with the literature.9,11
In summary, the results of this study have proved the
effectiveness of PDI with the use of modified
hematoporphyrin IX on the S mutans strain studied, indi-
cating that therapy with the use of this photosensitizer
associated with blue LED can be used to prevent WSLs
and tooth decay in the near future, because this pathol-
ogy is usually associated with the presence of S mutans
biofilm as a potential etiologic agent. New photosensi-
tizer concentrations of hematoporphyrin IX and other
doses may be tested to achieve the photodynamic inacti-
vation of the S mutans strain UA 159. The synthesis of
new and more efficient photosensitizers is an important
and interesting approach to improve results in PDI and
it is a potential field for new translational and clinical
research in the future.
CONCLUSIONS
The present results showed that hematoporphyrin IX
and modified hematoporphyrin IX associated with blue
LED exerted significant antimicrobial activity in
in vitro planktonic cultures. Regarding cultures of
S mutans in biofilm phase on metallic and
ceramic brackets, no effect was observed with the
February 2019  Vol 155  Issue 2 American Journal of Orthodontics and Dentofacial Orthopedics
Lacerda Rangel Esper et al
use of hematoporphyrin IX. However, modified
hematoporphyrin IX showed better results than
hematoporphyrin IX, indicating a promising use against
dental caries and WSL prevention.
ACKNOWLEDGMENTS
The authors thank CAPES (Coordenaç~ao de Aperfei-
çoamento de Pessoal de Nıvel Superior) for their finan-
cial support.
REFERENCES
1. Esper MA, Nicolau RA, Arisawa EA. The effect of two phototherapy
protocols on pain control in orthodontic procedure—a preliminary
clinical study. Lasers Med Sci 2011;26:657-63.
2. Krishnan V. Orthodontic pain: from causes to management—a
review. Eur J Orthod 2007;29:170-9.
3. Pandis N, Papaioannou W, Kontou E, Nakou M, Makou M, Eliades T.
Salivary Streptococcus mutans levels in patients with conventional
and self-ligating brackets. Eur J Orthod 2010;32:94-9.
4. Batoni G, Pardini M, Giannotti A, Ota F, Giuca MR, Gabriele M,
et al. Effect of removable orthodontic appliances on oral
colonisation by mutans streptococci in children. Eur J Oral Sci
2001;109:388-92.
5. Sukontapatipark W, el-Agroudi MA, Selliseth NJ, Thunold K,
Selvig KA. Bacterial colonization associated with fixed orthodontic
appliances. A scanning electron microscopy study. Eur J Orthod
2001;23:475-84.
6. Lovrov S, Hertrich K, Hirschfelder U. Enamel demineralization
during fixed orthodontic treatment—incidence and correlation to
various oral-hygiene parameters. J Orofac Orthop 2007;68:353-63.
7. Smiech-Slomkowska G, Jablonska-Zrobek J. The effect of oral
health education on dental plaque development and the level of
caries-related Streptococcus mutans and Lactobacillus spp. Eur
J Orthod 2007;29:157-60.
8. Uzuner FD, Kaygisiz E, Cankaya ZT. Effect of the bracket types on
microbial colonization and periodontal status. Angle Orthod 2014;
84:1062-7.
9. Jurela A, Repic D, Pejda S, Juric H, Vidakovic R, Matic I, et al. The
effect of two different bracket types on the salivary levels of
S mutans and S sobrinus in the early phase of orthodontic
treatment. Angle Orthod 2013;83:140-5.
10. Pandis N, Vlachopoulos K, Polychronopoulou A, Madianos P,
Eliades T. Periodontal condition of the mandibular anterior
dentition in patients with conventional and self-ligating brackets.
Orthod Craniofac Res 2008;11:211-5.
11. Papaioannou W, Gizani S, Nassika M, Kontou E, Nakou M.
Adhesion of Streptococcus mutans to different types of brackets.
Angle Orthod 2007;77:1090-5.
12. Gorelick L, Geiger AM, Gwinnett AJ. Incidence of white spot
formation after bonding and banding. Am J Orthod 1982;81:93-8.
13. Rosenbloom RG, Tinanoff N. Salivary Streptococcus mutans levels
in patients before, during, and after orthodontic treatment. Am J
Orthod Dentofacial Orthop 1991;100:35-7.
14. Eliades T, Eliades G, Brantley WA. Microbial attachment on
orthodontic appliances: I. Wettability and early pellicle formation on
bracket materials. Am J Orthod Dentofacial Orthop 1995;108:351-60.
15. Ogaard B. Prevalence of white spot lesions in 19-year-olds: a study
on untreated and orthodontically treated persons 5 years after
treatment. Am J Orthod Dentofacial Orthop 1989;96:423-7.
Lacerda Rangel Esper et al
16. Guzman-Armstrong S, Chalmers J, Warren JJ. Ask us. White spot
lesions: prevention and treatment. Am J Orthod Dentofacial
Orthop 2010;138:690-6.
17. Bahoum A, Bahije L, Zaoui F. [Enamel demineralization in
orthodontics. Systematic use of fluoride in prevention and
treatment]. Schweiz Monatsschr Zahnmed 2012;122:937-47.
18. O'Reilly MM, Featherstone JD. Demineralization and
remineralization around orthodontic appliances: an in vivo study.
Am J Orthod Dentofacial Orthop 1987;92:33-40.
19. Ogaard B, Rolla G, Arends J. Orthodontic appliances and enamel
demineralization. Part 1. Lesion development. Am J Orthod
Dentofacial Orthop 1988;94:68-73.
20. Featherstone JD. The science and practice of caries prevention. J
Am Dent Assoc 2000;131:887-99.
21. Alves KM, Goursand D, Zenobio EG, Cruz RA. Effectiveness of
procedures for the chemical-mechanical control of dental biofilm
in orthodontic patients. J Contemp Dent Pract 2010;11:41-8.
22. Lee YH, Park HW, Lee JH, Seo HW, Lee SY. The photodynamic
therapy on Streptococcus mutans biofilms using erythrosine and
dental halogen curing unit. Int J Oral Sci 2012;4:196-201.
23. Alves de Souza R, Borges de Araujo Magnani MB, Nouer DF,
Oliveira da Silva C, Klein MI, Sallum EA, et al. Periodontal
and microbiologic evaluation of 2 methods of archwire
ligation: ligature wires and elastomeric rings. Am J Orthod
Dentofacial Orthop 2008;134:506-12.
24. Birang R, Shahaboui M, Kiani S, Shadmehr E, Naghsh N. Effect of
nonsurgical periodontal treatment combined with diode laser or
photodynamic therapy on chronic periodontitis: a randomized
controlled split-mouth clinical trial. J Lasers Med Sci 2015;6:
112-9.
25. Kimura M, Miyajima K, Kojika M, Kono T, Kato H. Photodynamic
therapy (PDT) with chemotherapy for advanced lung cancer with
airway stenosis. Int J Mol Sci 2015;16:25466-75.
26. Pereira CA, Costa AC, Carreira CM, Junqueira JC, Jorge AO. Photo-
dynamic inactivation of Streptococcus mutans and Streptococcus
sanguinis biofilms in vitro. Lasers Med Sci 2013;28:859-64.
27. Konopka K, Goslinski T. Photodynamic therapy in dentistry. J Dent
Res 2007;86:694-707.
28. Tavares A, Carvalho CM, Faustino MA, Neves MG, Tome JP,
Tome AC, et al. Antimicrobial photodynamic therapy: study of
bacterial recovery viability and potential development of resistance
after treatment. Mar Drugs 2010;8:91-105.
29. DeRosa MC, Crutchley RJ. Photosensitized singlet oxygen and its
applications. Coord Chem Rev 2002;233-234:351-71.
30. Rossi LM, Silva PR, Vono LL, Fernandes AU, Tada DB, Baptista MS.
Protoporphyrin IX nanoparticle carrier: preparation, optical
properties, and singlet oxygen generation. Langmuir 2008;24:
12534-8.
31. Uchoa AF, Oliveira CS, Baptista MS. Relationship between
structure and photoactivity of porphyrins derived from
protoporphyrin IX. J Porphyr Phthalocyanines 2010;14:832-45.
32. Zanin IC, Goncalves RB, Junior AB, Hope CK, Pratten J. Suscepti-
bility of Streptococcus mutans biofilms to photodynamic therapy:
an in vitro study. J Antimicrob Chemother 2005;56:324-30.
33. Wilson BC, Patterson MS. The physics, biophysics and technology
of photodynamic therapy. Phys Med Biol 2008;53:R61-109.
34. Costa AC, Chibebe Junior J, Pereira CA, Machado AK, Beltrame
Junior M, Junqueira JC, et al. Susceptibility of planktonic cultures
of Streptococcus mutans to photodynamic therapy with a
light-emitting diode. Braz Oral Res 2010;24:413-8.
35. Habiboallah G, Mahdi Z, Mahbobeh NN, Mina ZJ, Sina F, Majid Z.
Bactericidal effect of visible light in the presence of erythrosine
on Porphyromonas gingivalis and Fusobacterium nucleatum
American Journal of Orthodontics and Dentofacial Orthopedics
253
compared with diode laser, an in vitro study. Laser Ther 2014;
23:263-71.
36. Wilson M. Lethal photosensitisation of oral bacteria and its
potential application in the photodynamic therapy of oral
infections. Photochem Photobiol Sci 2004;3:412-8.
37. Trindade FZ, Pavarina AC, Ribeiro AP, Bagnato VS, Vergani CE,
Costa CA. Toxicity of photodynamic therapy with LED associated
to Photogem: an in vivo study. Lasers Med Sci 2012;27:403-11.
38. Wainwright M, Crossley KB. Methylene blue—a therapeutic dye for
all seasons? J Chemother 2002;14:431-43.
39. Rossoni RD, Junqueira JC, Santos EL, Costa AC, Jorge AO.
Comparison of the efficacy of rose Bengal and erythrosin in
photodynamic therapy against Enterobacteriaceae. Lasers Med
Sci 2010;25:581-6.
40. Paschoal MA, Tonon CC, Spolidorio DM, Bagnato VS, Giusti JS,
Santos-Pinto L. Photodynamic potential of curcumin and blue
LED against Streptococcus mutans in a planktonic culture.
Photodiagnosis Photodyn Ther 2013;10:313-9.
41. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating
antimicrobial activity: a review. J Pharm Anal 2016;6:71-9.
42. Cieplik F, Buchalla W, Hellwig E, Al-Ahmad A, Hiller KA, Maisch T,
et al. Antimicrobial photodynamic therapy as an adjunct for
treatment of deep carious lesions—a systematic review.
Photodiagnosis Photodyn Ther 2017;18:54-62.
43. Nunez S, Ribeiro M, Garcez A. Terapia fotodin^amica
antimicrobiana na Odontologia. Rio de Janeiro: Elsevier; 2013.
44. Wood S, Metcalf D, Devine D, Robinson C. Erythrosine is a
potential photosensitizer for the photodynamic therapy of oral
plaque biofilms. J Antimicrob Chemother 2006;57:680-4.
45. Allaker RP, Douglas CW. Novel antimicrobial therapies for dental
plaque–related diseases. Int J Antimicrob Agents 2009;33:8-13.
46. Meisel P, Kocher T. Photodynamic therapy for periodontal dis-
eases: state of the art. J Photochem Photobiol B 2005;79:159-70.
47. Engelmann FM, Mayer I, Gabrielli DS, Toma HE, Kowaltowski AJ,
Araki K, et al. Interaction of cationic meso-porphyrins with
liposomes, mitochondria and erythrocytes. J Bioenerg Biomembr
2007;39:175-85.
48. Sudhakara Reddy R, Ramya K, Tatapudi R, Gudapati S, Sai
Madhavai N, Sai Kiran C. Photo dynamic therapy in oral diseases.
Int J Biol Med Res 2012;3:1875-83.
49. Paschoal MA, Moura CM, Jeremias F, Souza JF, Bagnato VS,
Giusti JS, et al. Longitudinal effect of curcumin–photodynamic
antimicrobial chemotherapy in adolescents during fixed orthodon-
tic treatment: a single-blind randomized clinical trial study. Lasers
Med Sci 2015;30:2059-65.
50. Mang TS, Tayal DP, Baier R. Photodynamic therapy as an
alternative treatment for disinfection of bacteria in oral biofilms.
Lasers Surg Med 2012;44:588-96.
51. Metcalf D, Robinson C, Devine D, Wood S. Enhancement of
erythrosine-mediated photodynamic therapy of Streptococcus
mutans biofilms by light fractionation. J Antimicrob Chemother
2006;58:190-2.
52. Nagata JY, Hioka N, Kimura E, Batistela VR, Terada RS,
Graciano AX, et al. Antibacterial photodynamic therapy for dental
caries: evaluation of the photosensitizers used and light source
properties. Photodiagnosis Photodyn Ther 2012;9:122-31.
53. Silva JR, Cardoso G, Maciel RR, de Souza NC. Morphological
alterations on Citrobacter freundii bacteria induced by erythrosine
dye and laser light. Lasers Med Sci 2015;30:469-73.
54. Ishiyama K, Nakamura K, Ikai H, Kanno T, Kohno M, Sasaki K, et al.
Bactericidal action of photogenerated singlet oxygen from
photosensitizers used in plaque disclosing agents. PLoS One
2012;7:e37871.
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